本文關(guān)鍵詞：轉(zhuǎn)錄因子誘餌策略中由載體表達的雙鏈寡核苷酸的功能研究　出處：《第四軍醫(yī)大學》2009年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 轉(zhuǎn)錄因子誘餌策略 ODN 相似序列 雙鏈RNA shRNA

【摘要】： 轉(zhuǎn)錄因子誘餌策略(TFD strategy,Transcription Factor Decoy strategy)是近年來研究和應用較廣的一種用來探索轉(zhuǎn)錄因子功能的策略。利用人工合成的含有某轉(zhuǎn)錄因子結(jié)合位點序列的小片段脫氧寡核苷酸雙鏈能與該轉(zhuǎn)錄因子正常的結(jié)合位點競爭結(jié)合轉(zhuǎn)錄因子的特性,達到封閉該轉(zhuǎn)錄因子活性的目的。近年來已有眾多利用TFD策略研究轉(zhuǎn)錄因子的報道,如NFκB、Sp1、NFAT等等。轉(zhuǎn)錄因子誘餌策略以其對轉(zhuǎn)錄因子封閉效果切實、功能分子為小分子等優(yōu)點被越來越廣泛地應用到分子生物學領(lǐng)域,并于近期初步用于臨床,取得了較好效果。 雖然轉(zhuǎn)錄因子誘餌策略有眾多優(yōu)點,但其缺陷同樣突出,其中以功能分子脫氧寡核苷酸ODN(oligodeoxynucleotide)的細胞核轉(zhuǎn)移效率過低最為明顯。TFD策略發(fā)揮作用需要ODN進入細胞核內(nèi)與轉(zhuǎn)錄因子結(jié)合,但未經(jīng)修飾的ODN入核后有大約90%的分子被溶酶體攝取降解,僅有10%左右的ODN能進入細胞核發(fā)揮作用。這對TFD策略的實際應用產(chǎn)生很大的影響。目前有數(shù)種方案用以解決這項難題,如引入核定位信號肽(NLS)交聯(lián)ODN以提高核轉(zhuǎn)移效率、對ODN進行修飾以提高其對溶酶體降解的抵抗力以及利用新型轉(zhuǎn)染試劑提高小分子(ODN)的轉(zhuǎn)染效率等。但這些方案或者成本過高,或者工藝復雜,未能被廣泛應用。因此尚需探索新的、更方便經(jīng)濟的方法。 我們在對莖-環(huán)小干擾RNA即shRNA的研究中發(fā)現(xiàn),其在細胞內(nèi)表達過程會出現(xiàn)一個階段,表達出的單鏈RNA分子會形成暫時性的局部雙鏈。我們設(shè)想,既然含有轉(zhuǎn)錄因子結(jié)合位點序列的ODN(系雙鏈DNA)具有相應的轉(zhuǎn)錄因子封閉功能,那么與某轉(zhuǎn)錄因子結(jié)合位點序列相似的雙鏈RNA是否也具有類似的封閉效應呢?如果猜想是正確的話,那我們就可以利用shRNA來表達這種雙鏈RNA,將載體包裝病毒,這樣既可以達到封閉的目的,又極大地提高了功能分子的核轉(zhuǎn)移效率。因此本課題將對此進行探討。 【目的】 1.明確能夠表達與某轉(zhuǎn)錄因子結(jié)合位點序列相似的雙鏈RNA的載體是否能夠封閉該轉(zhuǎn)錄因子的活性;2.探索這種載體發(fā)揮封閉功能的機制 【方法】 1.選擇在HEK293細胞中有表達的轉(zhuǎn)錄因子NFκB,構(gòu)建其相關(guān)載體,包括目的載體即表達上述雙鏈RNA的載體(vector-based ON, V-B ON)及其錯序?qū)φ誷crambled V-B ON,同時構(gòu)建NFκB的shRNA作為陽性對照1,合成未經(jīng)修飾的NFκB結(jié)合位點序列ODN作為陽性對照2及其錯序?qū)φ誷crambled ODN;2.將各種載體或小分子分別轉(zhuǎn)染HEK293細胞,MTT試驗檢測細胞生長狀態(tài)變化;流式細胞術(shù)檢測轉(zhuǎn)染后細胞受藥物作用后的凋亡情況;實時定量PCR及熒光素酶報告基因?qū)嶒灆z測轉(zhuǎn)染后細胞內(nèi)NFκB下游基因IL-8及MCP-1轉(zhuǎn)錄活性變化;3.隨機選擇在HEK293細胞中有表達的3種轉(zhuǎn)錄因子(FOS、AP-2α及RXRA),實時定量PCR檢測轉(zhuǎn)染后細胞內(nèi)這三種轉(zhuǎn)錄因子下游基因轉(zhuǎn)錄活性的變化以明確這種封閉效應是否廣泛存在;4.實時定量PCR檢測轉(zhuǎn)染后細胞內(nèi)NFκB、FOS、AP-2α及RXRA各轉(zhuǎn)錄因子本身轉(zhuǎn)錄活性的變化,以明確封閉效應是否系載體的RNAi作用所致,探索載體V-B ON發(fā)揮作用的機制。 【結(jié)果】 1.目的載體V-B ON能夠抑制HEK293細胞的生長,促進轉(zhuǎn)染后細胞凋亡我們在成功構(gòu)建各種載體的基礎(chǔ)上,將各載體及小分子ODN分別轉(zhuǎn)染HEK293細胞,分別在24小時、48小時及72小時做MTT試驗。結(jié)果證明,三個時段均出現(xiàn)相似的趨勢,即V-B ON與shRNA對細胞生長的相對抑制率相似(V-B ON:42.3±0.152%、57.2±0.612%、29.1±0.441%; shRNA:54.2±0.231%、64.3±0.246%、31.6±0.225%),而合成的小片段ODN對細胞生長的相對抑制率則較低(ODN:18.2±0.294%、21.1±0.546%、11.6±0.331%)。 按MTT試驗得出的結(jié)論,選擇48小時作為以下其他實驗的處理時間。各載體及小片段轉(zhuǎn)染細胞24小時后,向各組加依托伯苷(VP-16)至終濃度50umol/L。藥物處理24小時后收集細胞做流式細胞術(shù)。結(jié)果顯示,V-B ON與shRNA對轉(zhuǎn)染后細胞凋亡的促進程度即相對抑制率相似(V-B ON:42.2±0.437% vs shRNA:50.2±0.512%),而小片段ODN促進程度則較低(22.3±0.601%)。以上相對抑制率為各載體或分子對各自陰性對照的相對抑制率(V-B ON vs scrambled V-B ON;shRNA vs空白載體;ODN vs scrambled ODN)。 MTT試驗及流式細胞術(shù)均證明V-B ON能夠抑制HEK293細胞生長,促進其凋亡,效果與shRNA相似。 2. V-B ON能夠影響轉(zhuǎn)染后細胞內(nèi)NFκB、FOS、AP-2α及RXRA各因子下游基因轉(zhuǎn)錄 成功構(gòu)建了NFκB下游基因IL-8及MCP-1的熒光素酶報告基因載體。報告基因分析證明,轉(zhuǎn)染V-B ON后細胞內(nèi)IL-8及MCP-1的轉(zhuǎn)錄活性明顯較陰性對照組降低。 成功構(gòu)建FOS、AP-2α及RXRA等因子的相關(guān)載體(V-B ON、scrambledV-B ON及shRNA)。將NFκB、FOS、AP-2α及RXRA等因子的各載體或小分子分別轉(zhuǎn)染細胞,提取RNA,反轉(zhuǎn)錄并做實時定量PCR(SYBR Green)。結(jié)果顯示,各因子下游基因mRNA表達出現(xiàn)程度不一的抑制(轉(zhuǎn)錄因子抑制其表達者則是活性升高)。V-B ON與shRNA的抑制率相似,而小片段ODN效果較差。 3. V-B ON并不會影響其相應轉(zhuǎn)錄因子在轉(zhuǎn)染后細胞內(nèi)的轉(zhuǎn)錄活性,說明V-B ON至少并非通過對相應轉(zhuǎn)錄因子的RNA干擾來發(fā)揮作用 NFκB、FOS、AP-2α及RXRA等因子的各載體或小分子分別轉(zhuǎn)染細胞,提取RNA,反轉(zhuǎn)錄并做實時定量PCR,檢測四種轉(zhuǎn)錄因子本身轉(zhuǎn)錄活性的變化。結(jié)果顯示,與錯序?qū)φ障啾?V-B ON未表現(xiàn)出明顯的抑制效果,而shRNA對各分子活性的抑制則很明顯。小片段ODN對各分子活性亦無明顯影響。 【結(jié)論】 本研究首次提出能夠表達與某轉(zhuǎn)錄因子結(jié)合位點序列相似的雙鏈RNA的載體可能具有與轉(zhuǎn)錄因子誘餌策略中ODN相似的轉(zhuǎn)錄因子封閉功能的假說,提供了該類載體封閉相應轉(zhuǎn)錄因子活性的功能性證據(jù),并初步探討了其可能機制。這種載體很可能是通過其表達的序列上與某轉(zhuǎn)錄因子結(jié)合位點相似的雙鏈RNA發(fā)揮ODN樣作用的。這些發(fā)現(xiàn)有助于提高對RNAi及TFD策略的進一步認識,并可能為分子生物學提供新的有力工具。
[Abstract]:Transcription factor decoy (TFD strategy, Transcription Factor Decoy strategy) is a transcription factor used to explore the function of strategy research in recent years and is widely used. The use of synthetic containing a transcription factor binding site sequence of small fragments of double stranded oligonucleotides can binding sites of transcription factors and competitive binding characteristics of the normal transcription factor the reach to close the transcription factor activity. In recent years there have been numerous transcription factors using TFD strategy research reports, such as Sp1, NF kappa B, NFAT and so on. Transcription factor decoy on transcription factors and blocking effect of functional molecules into small molecules, etc. are increasingly widely used in molecular biology the field, and preliminarily used in the recent clinical, and achieved good results.
Although the transcription factor decoy strategy has many advantages, but its defect is also prominent, with the function of molecular ODN oligodeoxynucleotide (oligodeoxynucleotide) of the nuclear transfer of low efficiency is the most obvious.TFD strategies play a role in combination with transcription factor ODN into the nucleus, but without modification of the ODN into the nucleus after about 90% of the uptake of lysosomal molecules degradation, only about 10% of the ODN can translocate into nucleus. This greatly affect the practical application of the TFD strategy. Several schemes to solve this problem, such as the introduction of nuclear localization signal peptide (NLS) in order to improve the efficiency of nuclear transfer ODN crosslinking of ODN, modified to improve the lysosomal degradation the resistance and improve the utilization of new small molecule transfection reagent (ODN) transfection efficiency. But these solutions or the cost is too high, or complex technology, has not been widely used. It is still necessary to explore new, more convenient and economical ways.
We are on the stem loop of small interfering RNA shRNA study found that the expression of the emergence of a stage in the cell, single stranded RNA molecules expressed will form a temporary local double chain. We assume that since it contains transcription factor binding sites of ODN (double stranded DNA) is a transcription factor the corresponding sealing function, then combined with a transcription factor of double stranded RNA sequences similar whether it has closed to similar effects? If the conjecture is correct, then we can use shRNA to express the double stranded RNA, the carrier for viral packaging, this can achieve the purpose of sealing, and greatly improve the efficiency of nuclear transfer function molecules. So this topic will be discussed.
[Objective]
1., it is clear whether a double stranded RNA that is similar to a transcription factor binding site can block the activity of the transcription factor. 2., explore the mechanism of this vector to play a closed function.
[method]
The 1. is the expression of the transcription factor NF kappa B in HEK293 cells, construct the relevant carrier carrier including objective vector that is the expression of double stranded RNA (vector-based ON, V-B ON) and scrambled V-B ON wrong sequence control, and construction of NF kappa B shRNA as a positive control 1, the synthesis of unmodified NF B binding site sequence ODN as positive control and 2 wrong sequence control scrambled ODN; 2. kinds of carrier or small molecules were transfected into HEK293 cells, MTT cell growth state change test; flow cytometry to detect the apoptosis of cells after transfection after drug action; downstream changes in intracellular NF kappa B gene IL-8 and MCP-1 the transcriptional activity of real-time PCR and luciferase reporter assay after transfection; 3. randomly selected 3 transcription factor expression in HEK293 cells (FOS, AP-2 alpha and RXRA), real time quantitative PCR detection of transfected cells in the three turn Changes of downstream transcriptional activity of transcription factors to determine whether the sealing effect exists; 4. real time quantitative PCR detection after transfection of NF kappa B, FOS, AP-2 and RXRA all the changes of alpha transcription factor itself transcriptional activity, the effect of RNAi clear whether the sealing effect caused by the carrier, to explore the role of ON carrier V-B the mechanism.
[results]
1. destination vector V-B ON can inhibit the growth of HEK293 cells, promote apoptosis after transfection, we based on the successful construction of various vectors, each vector and small molecule ODN were transfected into HEK293 cells, respectively, in 24 hours, 48 hours and 72 hours to do the MTT test. The results prove that the three periods showed similar trends V-B, ON and shRNA on cell growth inhibition rate relatively similar (V-B ON:42.3 + 0.152%, 57.2 + 0.612%, 29.1 + 0.441%; shRNA:54.2 + 0.231%, 64.3 + 0.246%, 31.6 + 0.225%), while the small fragments of ODN on the synthesis of cell growth inhibition rate is relatively low (ODN:18.2 0.294%, 21.1 + 0.546%, 11.6 + 0.331%).
According to the MTT result, 48 hours of processing time following other experiments. After 24 hours of each carrier and small fragments of transfected cells, and relying on the primary glycoside to each of the groups (VP-16) to a final concentration of 50umol/L. drugs after 24 hours of treatment were collected as flow cytometry. The results showed that the relative inhibition rate, degree of enhancement similar to cell apoptosis after transfection with V-B ON shRNA (V-B ON:42.2 + 0.437% vs shRNA:50.2 + 0.512%), while the small fragments of ODN promote the degree is low (22.3 + 0.601%). The relative inhibition rate for each carrier or molecules on their negative control of ON vs scrambled V-B (the inhibition rate of V-B ON; shRNA vs ODN vs scrambled ODN blank vector;).
Both MTT test and flow cytometry showed that V-B ON could inhibit the growth of HEK293 cells and promote its apoptosis, and the effect was similar to that of shRNA.
2. V-B ON can affect the downstream gene transcription of NF kappa B, FOS, AP-2 alpha and RXRA factors after transfection
The luciferase reporter vector of NF and B downstream genes IL-8 and MCP-1 were successfully constructed. The analysis of the reporter gene showed that the transcriptional activity of IL-8 and MCP-1 decreased significantly compared with the negative control group after transfection of V-B ON.
The successful construction of FOS, AP-2 and RXRA related carrier alpha factor (V-B ON, scrambledV-B ON and shRNA). NF FOS, RNA K B, extraction of each carrier or small molecule AP-2 alpha and RXRA factor were transfected cells, reverse transcription and quantitative real-time PCR (SYBR Green). The results showed that the expression of mRNA downstream genes of each factor appears to inhibit the level of (transcription factor inhibiting its expression is increased the activity of inhibition of.V-B ON and shRNA) were similar, and the poor small fragments of ODN.
3. V-B ON does not affect the transcriptional activity of its corresponding transcription factors after transfection, indicating that V-B ON does not play a role in RNA interference at least.
NF FOS, RNA K B, extraction of each carrier or small molecule AP-2 alpha and RXRA factor were transfected cells, reverse transcription and quantitative real-time PCR, change detection of four kinds of transcription factor itself transcriptional activity. The results showed that, compared with the wrong sequence control, V-B ON did not show inhibitory effect is obvious. The inhibition of shRNA on the activity of a molecule is very obvious. There is no obvious effect on the activity of small fragments of ODN molecules.
[Conclusion]
This is the first study to propose expression combined with the vector of double stranded RNA sequences with a similar transcription factor and transcription factor ODN may have similar closed function in transcriptional factor decoy strategy hypothesis, provides the carrier closed functional evidence corresponding transcription factor activity, and to explore its possible mechanism. This vector is double stranded RNA binding sites may be similar to a transcription factor through the sequence of its expression play ODN like effect. These findings are helpful to improve the further understanding of the RNAi and TFD strategy, and may provide a new powerful tool for molecular biology.

【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R346

【引證文獻】

相關(guān)碩士學位論文 前1條

1 靳江;轉(zhuǎn)錄因子EGR-3在肝癌中的表達及意義[D];第四軍醫(yī)大學;2010年

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本文編號：1437321